Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
  • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/19—Cytokines; Lymphokines; Interferons
  • A61K38/20—Interleukins [IL]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/19—Cytokines; Lymphokines; Interferons
  • A61K38/21—Interferons [IFN]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
  • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
  • A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
  • A61K47/66—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid the modifying agent being a pre-targeting system involving a peptide or protein for targeting specific cells
  • A61K47/665—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid the modifying agent being a pre-targeting system involving a peptide or protein for targeting specific cells the pre-targeting system, clearing therapy or rescue therapy involving biotin-(strept) avidin systems
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
  • A61K9/0024—Solid, semi-solid or solidifying implants, which are implanted or injected in body tissue
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B82—NANOTECHNOLOGY
  • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
  • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Definitions

• the present invention relates to novel pharmaceutical formulations based on stable and fluid aqueous colloidal suspensions for the sustained release of proteinaceous active ingredients, namely interferons (IFN), as well as the active pharmaceutical agents. therapeutic applications of these formulations.
• IFN interferons
• These active pharmaceutical formulations concern both human and veterinary therapeutics.
• Interferons are glycoproteins belonging to the family of cytokines. They are biological mediators that, by binding to membrane receptors, trigger a pleiotropic cellular response. This results in antiviral, antiproliferative and immunomodulatory activity. Interferons have also been recognized as effective anti-tumor or anticancer agents. Interferon refers to all interferon fungi, such as alpha, beta or gamma interferons.
• IFN can be produced by genetic engineering.
• the sustained-release pharmaceutical formulations of TFN are subject to the need to reproduce at best in the patient a plasma concentration of IFN close to the value observed in the healthy subject. This objective is hampered by the short lifespan of IFNs in plasma, which makes it very difficult to inject them repeatedly.
• the plasma concentration of therapeutic protein then has a "sawtooth" profile characterized by high peaks of concentration and n ⁇ ttima very low concentration. Concentration peaks, well above the basal concentration in healthy subjects, have very strong adverse effects due to the high toxicity of IFNs. In addition, concentration levels are lower than the concentration required to have a therapeutic effect, resulting in poor therapeutic coverage of the patient and serious long-term side effects.
• the pharmaceutical formulation considered makes it possible to release the therapeutic protein over a prolonged period of time so as to limit the variations in plasma concentration at the same time. course of time.
• this active formulation should preferably satisfy the following specification, already known to those skilled in the art: 1 - prolonged release of one or more active and undenatured interferons (no modified), so that the plasma concentration is maintained at the therapeutic level, 2 - liquid form sufficiently fluid to be easily injectable and sterilizable by filtration on filters whose pore size is less than or equal to 0.2 microns, 3 - form stable liquid, 4 - biocompatibility and biodegradability, 5 - nontoxicity, 6 - non-immunogenicity, 7 - excellent local tolerance.
• 1 - prolonged release of one or more active and undenatured interferons (no modified) so that the plasma concentration is maintained at the therapeutic level
• 2 - liquid form sufficiently fluid to be easily injectable and sterilizable by filtration on filters whose pore size is less than or equal to 0.2 microns
• 3 - form stable liquid 4 - biocompatibility and biodegradability, 5 - nontoxicity, 6 - non-immunogenicity, 7 - excellent local tolerance
• the native therapeutic protein is modified by covalent grafting of one or more polymer chains or by covalent grafting of a protein such as human serum albumin (HSA).
• HSA human serum albumin
• the protein thus modified has a lower affinity for its receptors and its half-life time in the general circulation increases considerably. The amplitude of the concentration variation between peaks and troughs of plasma protein concentration is thus considerably reduced.
• NIRAFERON® PEG an interferon alpha 2b chemically modified by grafting a polyethylene glycol (PEG) chain of mass 12kD. This chemical modification results in an increase in half-life time in the patient from 6.8 to 33 hours.
• This solution comprises: (A) 10 to 80% by weight of a thermoplastic base polymer, biocompatible, biodegradable and insoluble in water or physiological fluids (for example PolyLactic and / or PolyGlycolic); (B) an organic solvent, such as N-methylpyrrolidone dispersing in physiological fluids; o (C) an active ingredient (AP); (D) and finally 1 to 50% by weight of a controlled release agent consisting of a block copolymer type PolyLacticG lycolique / PolyEthyleneGlycol.
• a thermoplastic base polymer biocompatible, biodegradable and insoluble in water or physiological fluids
• an organic solvent such as N-methylpyrrolidone dispersing in physiological fluids
• AP active ingredient
• D and finally 1 to 50% by weight of a controlled release agent consisting of a block copolymer type PolyLacticG lycolique / PolyEthyleneGlycol.
• (B) disperses or dissipates in the physiological fluids.
• (A) forms an encapsulant implant (C) which is not covalently bound to either (A) or (D) and which is then slowly released in vivo.
• the main disadvantage of this technique is to use an organic solvent (B), potentially denaturing for PA (C) (eg therapeutic proteins) and toxic for the patient.
• PA eg therapeutic proteins
• the in vivo hydrolysis of the polymer (A) generates an acid which can lead to local tolerance problems.
• PCT applications WO-A-99/18142 and WO-A-00/18821 relate to aqueous solutions of polymers which contain a PA in dissolved or colloidal form, which can be administered to warm-blooded animals, in particular by injection, and which form a deposit of PA (eg insulin) gelled in vivo, because the physiological temperature is higher than their gelation temperature. The gel thus formed releases the PA for a prolonged period.
• PA eg insulin
• the liquid / gel transformation temperatures of these triblock polymers are, for example, 36, 34, 30 and 26 ° C.
• This formulation is intended for local administration in a target organ (eg eye).
• the aggregation active ingredient that is formed in situ allows the slow release of the active ingredient in the target organ.
• hGH human growth hormone e
• the biocompatible polymer is, for example, a poly (lactide), a poly (glycolide) or a poly (lactide-co-glycolide) copolymer.
• the composition is, for example, in the form of a suspension of microspheres in a solution of sodium carboxymethylcellulose.
• This approach has several disadvantages: firstly, during the microsphere manufacturing process, the protein is contacted with potentially denaturing organic solvents. In addition, the microspheres are of a high size (1 to 1000 microns), which constitutes a constraint in terms of injection and easy sterilization on filters. Finally, local tolerance problems can arise during the in situ hydrolysis of the polymer.
• sustained-release forms of therapeutic protein consisting of liquid suspensions of nanoparticles loaded with proteins. These allowed the administration of the native protein in a liquid formulation of low viscosity.
• the nanoparticulate sustained release suspension consists of liposome suspensions in which the unmodified native therapeutic protein is encapsulated. After injection, the protein is progressively released from the liposomes, which prolongs the time of presence of the protein in the general circulation.
• Frossen et al. Describe in the article Cancer Res. 43 p 546, 1983 Encapsulation of anti-neoplastic agents in liposomes to increase therapeutic efficacy.
• Liposom e Company Inc in its US-B-5 399 331 patent proposes to improve the in vitro release time of interferon 2 by covalently grafting it to the liposome. We then fall back into the first "modified protein" approach mentioned above.
• Flamel Technologies has proposed a second prolonged release route in which the therapeutic protein is associated with nanoparticles. a water-soluble polymer "hydrophobic modified", that is to say, modified by grafting hydrophobic groups.
• This polymer is chosen, in particular, from polyamino acids (polyglutamates or polyaspartates) carrying hydrophobic grafts.
• polyamino acids polyglutamates or polyaspartates
• hydrophobic modified polymers One of the notable interests of these hydrophobic modified polymers is to self-assemble spontaneously in water to form nanoparticles.
• Another advantage of these systems is that the therapeutic proteins or peptides associate spontaneously with the nanoparticles of hydrophobic modified polymers, this combination is non-covalent and is carried out without using a surfactant or a potentially denaturing transformation process. This is not an encapsulation of the protein in a microsphere as disclosed in US-B-6,500,448 and US-A-2003/0133980.
• these nanoparticles of hydrophobic modified copolyamino acids spontaneously adsorb proteins in solut ion, without chemically modifying or denaturing them and without subjecting them to aggressive processing steps of the "emulsification” and "solvent evaporation” type.
• the formulations can be stored in liquid form or in lyophilized form. After injection, for example subcutaneously, these suspensions of protein-loaded nanoparticles progressively release the undenatured and bioactive protein in vivo.
• Such non-covalent combinations of active ingredient (PA) protein / poly [Glu] or poly [Asp] are disclosed in the patent application WO-A-00/30618.
• This application notably describes colloidal suspensions of pH 7.4 comprising combinations of human insulin with nanoparticles of "hydrophobic modified” polyglutamate.
• the table below gives an account of the "hydrophobic modified” polyamino acids used and the levels of association obtained in the examples of WO-A-00/30618.
• colloidal suspensions contain 1.4 mg / ml of insulin and 10 mg / ml of "hydrophobic modified" polyamino acid. It is apparent from FIG. 1 of WO-A-00/30618 that the in vivo release time of the insulin vectorized by the suspensions mentioned above is 12 h. This release duration would benefit to be increased. Thus, even if this PCT application already represents considerable progress, its technical content can still be optimized in view of the specifications set out above and especially with regard to the lengthening of the in vivo release time of interferons.
• amphiphilic "hydrophobic modified" polyamino acids according to the French patent application No. 02 07008 comprise aspartic units and / or glutamic units, carrying hydrophobic grafts comprising at least one alpha-tocopherol unit, eg: (polyglutamate or polyaspartate grafted with alpha tocopherol of synthetic or natural origin).
• This unpublished application specifically discloses a colloidal suspension which contains nanoparticles formed by polymer / active protein combinations and which is obtained by mixing 1 mg of a polyglutamate grafted with alpha-tocopherol and 7 mg of insulin in 1 ml. of water, at pH 7.0.
• the amphiphilic "hydrophilic modified" polyamino acids according to the French patent application No. 02 09670 comprise aspartic units and or glutamic units, carrying hydrophobic grafts comprising at least one hydrophobic unit and connected to the aspartic and or glutamic units via a ball joint containing two amide functions, and more specifically via a "spacer" of the lysine or ornithine type.
• 03 50190 comprise aspartic units and / or glutamic units, some of which carry at least one graft connected to an aspartic or glutamic unit, via a "spacer""aminoacid” based on Leu, and / or ILeu, and or Val, and / or Phe, a C6-C30 hydrophobic group being connected by an ester bond to the "spacer".
• This unpublished application specifically discloses a colloidal suspension which contains nanoparticles formed by polymer / active protein combinations and which is obtained by mixing an aqueous solution containing 10 mg of a grafted polyglutamate with a graft -Leu-OC8, -Val-OC12 or -Nal-cholesteryl and 200 IU insulin (7.4 mg) per milliliter of water at pH 7.4.
• French Patent Application No. 01 50641 discloses linear, amphiphilic, anionic homopolyamino acids, comprising aspartic units or glutamic units and whose ends carry hydrophobic groups containing from 8 to 30 carbon atoms.
• the "hydrophobic modified" telechelic homopolyamino acids are for example a poly [GluONa] with PheOC18 / C18 ends or a poly [GluONa] with PheOC18 / alpha-tocopherol ends.
• This unpublished application also describes a colloidal suspension which contains nanoparticles formed by polymer / active protein combinations and which is obtained by mixing 10 mg of one of the abovementioned polymers and 200 IU of insulin (7.4 mg) per milliliter. of water, at pH 7.4. The duration of in vivo release of insulin "vectorized" by the suspensions according to these unpublished requests, would benefit from being increased.
• Another essential objective of the invention is to propose a liquid pharmaceutical formulation with sustained release of interferon (s) in vivo, which is sufficiently fluid to be easily injectable and sterilizable by filtration on filters whose pore size is less than or equal to 0.2 microns.
• Another essential objective of the invention is to provide a liquid pharmaceutical formulation with sustained release of interferon (s) in vivo, which is stable to preservation both physico-chemical and biological.
• the invention is to propose a liquid pharmaceutical formulation with sustained release of interferon (s) in vivo, which has at least one of the following properties: biocompatibility, biodegradability, atoxicity, good local tolerance
• Another essential objective of the invention is to provide a pharmaceutical formulation for slow sustained release of interferon (s) in vivo, this formulation being a low viscosity aqueous colloidal suspension comprising submicron particles of polymer PO self-associated with at least one interferon (s), the polymer PO being a biodegradable, water-soluble polymer carrying hydrophobic groups
• another essential objective of the inv Invention is to propose a slow sustained release pharmaceutical formulation of interferon (s) in vivo, this formulation being an aqueous colloidal suspension of low viscosity comprising submicron particles of polymer PO self-associated with at least one interferon, the polymer PO being for example, a polyamino acid formed by aspartic units and / or glutamic units, at least a part of these units
• Another essential object of the invention is to propose derived products and / or precursors of the formulation referred to in the abovementioned objectives. It is in particular merit of the Applicant to have developed liquid aqueous pharmaceutical formulations of low viscosity at physiological temperature, which, surprisingly, form a gelled deposit in vivo after easy parenteral administration in humans or mammals with warm blood, the formation of this deposit is not triggered by a change in pH or temperature during parenteral injection, nor by the dispersion of organic solvent in the physiological medium. The gelled deposit thus formed significantly increases the in vivo release time of the BFN.
• the invention relates to a liquid pharmaceutical formulation for the sustained release of interferon (s), this formulation comprising a colloidal suspension, aqueous, of low viscosity, based on submicron particles of polymer (PO ) biodegradable, water-soluble and hydrophobic group-bearing (GH), said particles being non-covalently associated with at least one interferon and optionally with at least one other active ingredient (AP), characterized in that: suspension is essentially constituted by water, in that it is capable of being injected parenterally and then forming a gelled deposit in vivo, this gelled deposit formation: o being, on the one hand, at least part caused by at least one physiological protein present in vivo, and allowing, on the other hand, to prolong and control the release time of AP in vivo, beyond 24 hours after administration, * in that it is liquid under the conditions of injection, and in that it is also liquid at physiological temperature and / or pH, and / or in the presence of: physiological electrolyte at
• this gelation in vivo does not result from a change in pH and / or temperature, nor an in vivo dispersion of one or more organic solvents possibly contained in the injected formulation.
• physiological proteins present in vivo in physiological concentrations allow the aggregation of the PO nanoparticles associated with at least one interferon.
• Such gelling takes place, for example, in one or more hours, 24 hours, 48 hours or 72 hours, among others.
• the gelled deposit obtained after parenteral injection of the formulation allows an interesting prolongation of the duration of release of the protein as well as a reduction of the plasma concentration peak of interferon (s).
• the concentration of [PO] is such that it forms a deposition g eluted in vivo, after parenteral injection.
• the invention relates to a liquid pharmaceutical formulation for the sustained release of active principle (s).
• this formulation o being liquid in ambient atmosphere, o also being liquid at physiological temperature and / or pH and / or in the presence of: * physiological electrolyte at physiological concentration, * and / or at least one surfactant, o and comprising a colloidal suspension, aqueous, low viscosity, based on submicron particles of biodegradable PO polymer, water-soluble and bearing hydrophobic groups GH, said particles being non-covalently associated with at least one inter-feron (and optionally at least one other active ingredient) and the dispersing medium of the suspension consisting essentially of water, characterized in that its concentration of [PO] is set at a sufficiently high value to allow the formation of gelled deposit in vitro, after parenteral injection, in the presence of at least one protein .
• the liquid pharmaceutical formulation according to the invention is characterized in that its concentration of [PO] is such that: - [PO]> 0.9.C1, • preferably 20.C1>[PO]> C1, and more preferably 10.C1>[PO]> Cl with Cl representing the concentration of "induced gelation" of the PO particles as measured in a GI test.
• the gelled deposit obtained after parenteral injection of the formulation allows an interesting prolongation of the duration of release of the protein as well as a reduction of the plasma concentration peak of interferon (s).
• the AP release time is significantly increased over that of the prior art formulations, particularly those described in published PCT patent application WO-A-00/30618 and unpublished French patent applications.
• interferons are different from unmodified interferons or modified interferons, for example by grafting one or more polyoxyethylene groups.
• proteins of the inter feron family there may be mentioned: IFN alpha, IFN beta and IFN gamma.
• the associated supramolecular polymeric PO arrangements or not to at least one interferon and, optionally to at least one other active ingredient will be indifferently referred to as "submicron particles” or “nanoparticles”.
• these formulations are liquid, that is to say advantageously have a very low viscosity, which makes their injection easy. They gel only in vivo.
• the qualifiers "liquid”, “low” or “very low viscosity” correspond, advantageously, to a dynamic viscosity at 20 ° C. of less than or equal to 5 Pa ⁇ s.
• the reference measurement for the viscosity can be carried out, for example, at 20 ° C. using an AR1000 rheometer (TA Instruments) equipped with a cone-plane geometry (4 cm, 2 °).
• the viscosity of the formulations according to the invention may be, for the exemp, between 1.10" 3 and 5 Pa.s, preferably between 1.10 "and 3 0.8 Pa.s and even more preferably, between 1.10 "and 0.5 3 Pa.s.
• This low viscosity makes the formulations of the invention not only easily injectable parenterally, in particular intramuscularly or subcutaneously, inter alia, but also sterilizable easily and at a lower cost by filtration on 0.2 sterilization filters. ⁇ m of pore size.
• the formulation according to the invention is preferably an aqueous colloidal suspensio n of nanoparticles associated with one or more interferons and optionally one or more other PAs.
• the dispersive medium of this suspension is essentially formed by water. In practice, this water represents, for example, at least 50% by weight relative to the total weight of the formulation.
• the term "protein” refers to both a protein and a peptide. This protein or peptide may or may not be modified, for example by grafting one or more polyoxyethylene groups.
• physiological proteins is intended to mean the endogenous proteins and / or peptides of warm-blooded mammals present at the injection site.
• physiological temperature is intended to mean the physiological temperature of the warm-blooded mammals, namely, for example, approximately 37 ° -42 ° C.
• physiological pH is meant, in the sense of the invention, a pH for example between 6 and 7.6.
• the term “gel” means a semisolid state in which the liquid formulation according to the invention is converted, and this spontaneously by the mere presence of physiological protein (s), without essential intervention of the physiological pH and / or the physiological temperature and / or the presence of a physiological electrolyte (Ca ++ eg) and / or the dispersion (or dissipation) in vivo of an organic solvent possibly present in the injected formulation.
• physiological electrolyte is meant, within the meaning of the invention, any electrolyte element (for example Ca 44 ions) present in warm-blooded mammals.
• physiological concentration is meant, within the meaning of the invention, any physiological concentration encountered in warm-blooded mammals, for the physiological medium in question.
• the formulations according to the invention are nontoxic, well tolerated locally and stable. It is also the merit of the inventors to have developed an in vitro GI test for selecting a population of the preferred formulations according to the invention and to determine the appropriate concentrations of PO in the formulations.
• the GI test for measuring the gelation concentration Cl is a reference test that makes it possible to define the critical concentration Cl, hereinafter referred to as the Cl induced gelation concentration, which characterizes each colloidal formulation according to the invention.
• the GI test for determining the induced gelling concentration C1 is the following: In order to determine the concentration Cl, colloidal formulations of varying concentrations of amphiphilic polymer according to the invention and of constant concentration of therapeutic protein are prepared.
• the viscoelasticity measurements are carried out on a rheometer TA instrument s AR 1000, equipped with a cone-plane geometry (diameter 4cm and angle 1.59). A deformation of 0.01 rad, located in the field of linear viscoelasticity, is imposed sinusoidally over a frequency range between 0.1 and 300 rad / s. The temperature of the sample is kept constant at 20 ° C by means of a Peltier cell.
• the frequency spectra of the elastic modulus G 'and of the viscous or loss modulus, G " make it possible to define the characteristic relaxation time Tr defined here as the inverse of the frequency at which the elastic modulus G' crosses the viscous modulus G
• Tr defined here as the inverse of the frequency at which the elastic modulus G' crosses the viscous modulus G
• association or “associate” used to qualify the relations between one or more active ingredients and polymers PO (for example polyamino acids), mean in particular that the active principle (s) are (are) bound to the PO polymer (s) [for example the polyamino acid (s)] by a non-covalent bond, for example by electrostatic and / or hydrophobic interaction and / or hydrogen bonding and / or steric hindrance.
• the PO polymers according to the invention are biodegradable polymers, water-soluble and bearing hydrophobic groups GH.
• the hydrophobic groups may be in reduced number with respect to the rest of the chain and may be located laterally to the chain or intercalated in the chain, and may be randomly distributed (random copolymer) or distributed in the form of sequences or grafts ( block copolymers or block copolymers).
• the hydrophobic modified PO polymers may be chosen from the group comprising amphiphilic copolamino acids, polysaccharides, preferably in the subgroup including pullulans and / or chitosans and / or mucopolysaccharides, gelatins or their mixtures.
• PO is selected from amphiphilic copolyamino acids.
• polyamino acid covers both the oligomino acids comprising from 2 to 20 "amino acid” units and the polyamino acids comprising more than 20 "amino acid” units.
• the polyamino acids according to the present invention are oligomers or homopolymers comprising glutamic or aspartic acid repeating units or copolymers comprising a mixture of these two types of "amino acid” units.
• the units considered in these polymers are amino acids having the D or L or D / L configuration and are linked by their alpha or gamma positions for the glutamate or glutamic unit and alpha or beta for the aspartic or aspartate unit.
• the preferred "amino acid" units of the main polyamino acid chain are those having the L-configuration and an alpha-type bond.
• the polymer PO is a polyamino acid formed by aspartic units and / or glutamic units, at least a portion of these units carrying scions comprising at least one hydrophobic group GH.
• These polyamino acids are in particular of the type described in PCT patent application WO-A-00/30618.
• the PO (s) of the formulation are defined by the following general formula (I):
• R 1 represents H, linear C 2 -C 10 or branched C 3 -C 10 alkyl, benzyl, a terminal amino acid unit or -R 4 - [GH];
• R 2 represents an H, a C2 to C10 or branched C3 to C10 linear acyl group, a pyroglutamate or -R 4 - [GH];
• R 3 is an H or a cationic entity, preferably selected from the group comprising: metal cations advantageously chosen from the subgroup including: sodium, potassium calcium, magnesium, organic cations advantageously selected in the sub-group comprising: • amine-based cations, • oligoamine-based cations, • polya-based cations (polyeylenimine being particularly preferred), • acid-based cations (s) amine (s) advantageously chosen from the class comprising cations based on lysine or arginine, or cationic polyamino acids advantageously chosen from the subgroup comprising
• 1 GH represents a hydrophobic group
• R 30 is a C2 to C6 linear alkyl group
• R 3 ' is an H or a cationic entity, preferably selected from the group consisting of: - the metal cations advantageously chosen in the subgroup including: sodium, potassium, calcium, magnesium, - the cations organic compounds which are advantageously chosen from the sub-group comprising: • amine-based cations, • oligoamine-based cations, • polyamine-based cations (polyethyleneimine being particularly preferred), • cations based on amino acid (s) advantageously chosen from the class comprising cations based on lysine or arginine, or cationic polyamino acids advantageously chosen from the subgroup comprising polylysine or oligolysine,
• R 50 is a C2-C6 alkyl, dialkoxy or diamine group; R represents a direct bond or a "spacer" based on 1 to 4 amino acid units; A is independently a radical -CH 2 - (aspartic unit) or -CH 2 -CH 2 - (glutamic unit); B n '+ m' or n "is defined as the degree of polymerization and varies from 10 to 1000, preferably from 50 to 300.
• the n groups GH of the PO each represent each of them a monovalent radical of following formula:
• R 5 is methyl (alanine), isopropyl (valine), isobutyl (ucin), secbutyl (isoleucine), benzyl (phenylalanine);
• - R 6 represents a hydrophobic radical containing from 6 to 30 carbon atoms; - 1 varies from 0 to 6.
• all or part of the hydrophobic groups R 6 of the PO are independently selected from the group of radicals comprising: a linear or branched alkoxy containing from 6 to 30 carbon atoms and may comprise at least one heteroatom (preferably O and or N and / or S) or at least one unsaturation, "an alkoxy having 6 to 30 carbon atoms and having one or more annealed carbocycles and optionally containing at least one unsaturation and / or at least one heteroatom (preferably O and or N and / or S), an alkoxyaryl or an aryloxyalkyl of 7 to 30 carbon atoms and which may comprise at least one unsaturation and / or at least one a hetero-atom (preferably O and or N and / or S).
• PO is derived from an alcoholic precursor selected from the group consisting of: octanol, dodecanol, tetradecanol, hexadecanol, octadecanol, oleyl alcohol, tocopherol or cholesterol.
• the main chains of the polyamino acids are home-polymers of alpha-L-glutamate or alpha-L-glutamic acid.
• the main chains of the polyamino acids are homopolymers of alpha-L-aspartate or alpha-L-aspartic acid.
• the main chains of the polyamino acids are copolymers of alpha-L-aspartate / alpha-L-glutamate or alpha-L-aspartic / alpha-L-glutamic acid.
• the distribution of the aspartic and / or glutamic units of the main polyamino acid chain of the PO is such that the polymer thus constituted is either random, of the block type or of the multiblock type.
• the PO used in the formulation according to the invention has a molar mass which is between 2,000 and 100,000 g / mol, and preferably between 5,000 and 40,000 g / mol.
• the hydrophobic radical R 6 of the PO graft is derived from an alcoholic precursor formed by tocopherol: 1% ⁇ [n / (n + m)] ⁇ 100 ⁇ 10% of Preferably 3.5% ⁇ [n / (n + m)] ⁇ 100 ⁇ 7.5% 4 n + m varies from 100 to 400, preferably from 120 to 300.
• the hydrophobic radical 6 of the PO graft is derived from an alcoholic precursor formed by cholesterol: 41% ⁇ [n / (n + m)] x 100 ⁇ 10%, preferably 3.5% ⁇ [n / ( n + m)] x 100 ⁇ 6.5%> n + m varies from 100 to 400, preferably from 120 to 300.
• the concentration in polymer [PO] is between 15 and 50 mg / ml
• the PO of the formulation according to the invention carries at least one graft of polyalkylene-glycol type bonded to a glutamate and / or aspartate unit.
• this graft is of polyalkylene glycol type and is of formula (N) below.
• the polyalkylene glycol is, for example, a polyethylene glycol.It is desirable, according to the invention, for the molar percentage of grafting of the polyalkylene glycol to vary from 10 to 1000, preferably from 50 to 300.
• PO polyamino acids are also extremely interesting, because at an adjustable grafting rate, they are dispersed in water at pH 7.4 (for example with a phosphate buffer) to give suspensions
• active principles that are interferons or other PAs chosen from among proteins, peptides or small molecules can spontaneously associate with nanoparticles comprising these PO-polyamino acids.
• polyamino base contain carboxyl groups which are either neutral (COOH form) or ionized (COO anion "), depending on pH and composition. For this reason, the solubility in an aqueous phase is directly a function of the level of free COOH of the PO (not grafted by the hydrophobic unit) and the pH.
• the countercation may be a metal cation such as sodium, calcium or magnesium, or an organic cation such as triethanolamine, tris (hydroxymethyl) aminomethane or a polyamine such as pol yethyleneimine.
• PO polyamino acid type may be used in the formulation of the invention are, for example, obtained by methods known to those skilled in the art.
• the random polyamino acids can be obtained by grafting the hydrophobic gr, previously functionalized by the "spacer", directly onto the polymer by a conventional coupling reaction.
• the block or multiblock polyamino acid POs can be obtained by sequential polymerization of the corresponding N-carboxy-amino acid anhydrides (NCA).
• a polyamino acid, homopolyglutamate, homopolyaspartate or a glutamate / aspartate, block, multiblock or random copolymer is prepared according to conventional methods.
• N-carboxy-amino acid anhydrides NCA
• the most common technique is based on the polymerization of N-carboxy-amino acid anhydrides (NCA), described, for example, in the article "Biopolymers, 1976, 15, 1869 and in HR Kricheldorf's "Alpha-Aminoacid-N-carboxy Anhydride and related Heterocycles" Springer Verlag (1987)
• NCA derivatives are preferably NCA-O-Me, NCA-O-Et or NCA derivatives.
• Coupling of the graft with an acidic function of the polymer is easily achieved by reacting the polyamino acid in the presence of a carbodiimide as a coupling agent and optionally a catalyst such as 4-dimethylamino-pyridine and in a suitable solvent such as dimethylformamide (DMF). N-methyl pyrrolidone (NMP) or dimethyl sulfoxide (DMSO).
• a carbodiimide is, for example, dicyclohexylcarbodiimide or dnsopropylcarbodiimide.
• the degree of grafting is chemically controlled by the stoichiometry of the constituents and reactants or the reaction time.
• Hydrophobic grafts functionalized by a "spacer" are obtained by conventional peptide coupling or by direct condensation by acid catalysis. These techniques are well known to those skilled in the art.
• NCA derivatives previously synthesized with the hydrophobic graft are used!
• the NCA-hydrophobic derivative is copolymerized with the NCA-O-Benzyl and then the benzyl groups are selectively removed by hydrolysis.
• the synthesis of PO polyamino acids preferably leads to aqueous suspensions of PO nanoparticles.
• Such suspensions can be converted into powders of PO nanoparticles by drying, as appropriate and known to those skilled in the art, such as, for example: heating (oven, etc.), evacuation, use of desiccants , lyophilization, atomization.
• These nanoparticles of PO, in suspensions or in powder form form a raw material for the preparation of the formulations according to the invention.
• the formulations according to the invention result from the non-covalent combination of nanoparticles based on at least one PO and at least one PA, in an aqueous liquid medium.
• the PO and or interferon (s) may be in solid form (preferably powder) and / or in liquid form (preferably aqueous colloidal suspension).
• the interferon (s) / PO combination means, within the meaning of the present disclosure, that the interferon (s) is (are) associated with the PO polymer (s) [eg one or more polyamino acids (s) )] by one or more bonds other than one (or more) chemical bond (s) covalent (s).
• the techniques for associating one or more interleukins with the POs according to the invention are described in particular in the patent application WO-A-00/30618.
• the invention therefore also relates to a process for preparing the aforementioned formulation.
• this method is characterized in that it essentially consists in: implementing a colloidal suspension of nanoparticles of at least one PO, mixing this colloidal suspension of PO nanoparticles with at least an interferon (and one or more other possible active principle (s)), preferably in aqueous solution, to optionally add at least one excipient, 4 if necessary to adjust the pH and / or the osmolarity and, if necessary 4 filtering the suspension thus obtained.
• the interferon (and one or more other possible active principle (s)) is in the form of a suspension or an aqueous solution for mixing with the colloidal suspension of PO nanoparticles.
• this process is characterized in that it essentially consists in: implementing a powder of at least one PO polymer, 4 to mix this powder with a suspension or aqueous solution of at least one interferon (and one or more other active principle (s)), preferably in aqueous solution, 4 to optionally add at least one excipient, 4 if necessary to adjust the pH and / or the osmolarity and possibly to filter the suspension thus obtained.
• the formulations thus obtained can also be put in the form of gels, powder or film by conventional methods known to those skilled in the art, such as concentration by diafiltration or evaporation, coating, atomization or lyophilization, among others. These methods may possibly be combined.
• this third mode consisting essentially of: 4 to implement a powder resulting from the drying of the liquid formulation according to the invention; the invention as defined above, 4 to mix this powder with an aqueous liquid medium, preferably with stirring, 4 to optionally add at least one excipient, 4 if necessary to adjust the pH and or the osmolarity and, ••> optionally to filter the suspension thus obtained.
• the excipients which may be added are, for example, microbials, buffers, antioxidants, agents for adjusting isotonicity which are known to those skilled in the art.
• its mass fraction in mterleimjine (s) not associated with submicron particles [interleukin (s) not associated (s)] in% by weight is such that: o [interferon (s) not associated (s)] ⁇ 1 o preferably [interferon (s) not associated (s)] ⁇ 0.5.
• the preferred interferon is alpha interferon.
• the invention encompasses any derivative product obtained from the liquid formulation according to the invention as defined above and comprising submicron particles, formed by non-covalent PO / interferon combinations as defined above. .
• these derived products may in particular be constituted by powders, gels, implants or films, among others.
• the invention relates to any precursor of the injectable liquid formulation as defined above.
• the invention also relates to a process for preparing a powder derived from the formulation as defined above, this process being characterized in that said powder is obtained by drying the formulation as defined above.
• the formulation according to the invention is preferably pharmaceutical, without excluding cosmetic, dietetic or phytosanitary formulations comprising at least one PO as defined above and at least one interferon and optionally at least one other active ingredient.
• the optional additional active principle other than an interferon may be a protein, a glycoprotein, a protein linked to one or more polyalkylene glycol chains [preferably Polyethylene glycol (PEG): "PEG-protected protein”), a polysaccharide, a liposaccharide, an oligonucleotide, a polynucleotide or a peptide.
• PEG Polyethylene glycol
• This additional active ingredient may be selected from hemoglobins, cytochromes, albumins, interferons, cytokines, antigens, antibodies, erythropoietin, insulin, growth hormones, factors VIII and IX, factors stimulants of hematopoiesis or their mixtures.
• this additional active principle is a "small" hydrophobic, hydrophilic or amphiphilic organic molecule, for example peptides such as leuprolide or cyclosporin or small molecules such as those belonging to the family of anthracyclines, taxoids or Camp Tothecins and their mixtures.
• the formulation according to the invention may in particular be injected parenterally, subcutaneously, intramuscularly, intradermally, intraperitoneally, intracerebrally or into a tumor.
• the formulation according to the invention can also be administered orally, nasally, vaginally, ocularly or buccally.
• the formulation is intended for the preparation of medicaments, in particular for parenteral, subcutaneous, intramuscular, intradermal, intraperitoneal, intracerebral or tumoral administration, or even by oral, nasal, vaginal or ocular route.
• the formulation according to the invention is preferably pharmaceutical, this does not exclude cosmetic formulations, dietary or phytosanitary comprising at least one PO as defined above and at least one corresponding active ingredient.
• the invention is directed to a process for the preparation of medicaments, in particular for parenteral, subcutaneous, intramuscular, intradermal, intraperitoneal, intracerebral or tumor administration, or even orally, nasally, vaginally or vaginally.
• ocular characterized in that it consists essentially in implementing at least one above-defined formulation and / or any derivative product and / or any precursor of said formulation.
• the invention also relates to a method of therapeutic treatment essentially consisting in administering the formulation as described herein, parenterally, subcutaneously, intramuscularly, intradermally, intraperitoneally, intracerebrally or in a tumor, or even orally. , nasal, vaginal or ocular.
• this method of therapeutic treatment essentially consists in administering the formulation as described above by parenteral, subcutaneous, intramuscular, intradermal, intraperitoneal, intracerebral or tumor injection, preferably so as to it forms a gelled / crosslinked deposit on the injection site.
• FIG. 1 Plasma concentration curves of IFN (picogram / ml) obtained in the dog after subcutaneous injection • of the IFN (A) formulation according to the invention (Examples 9 & 10): (curve - M-M- • and of the control IFN (D) formulation outside the invention (Example 10): (-A- A- curve, as a function of time T in hours and at an IFN dose of 60 ⁇ g kg.
• Example 3 Preparation of 30 ml of a formulation of interferon alpha 2b (IFN) based on the polymer P6.
• IFN interferon alpha 2b
• the polymer concentration is adjusted to 45 mg / ml by addition of a sterile aqueous solution of 0.15M NaCl.
• the polymer solution is then filtered through a pore size filter of between 0.8 and 0.2 microns and stored at 4 ° C.
• Example 4 Preparation of a long-acting interferon (IFN) formulation according to the present invention, based on one of the polymers P1 to P5.
• IFN interferon
• the preparation is carried out as in Example 3, firstly preparing a colloidal polymer solution at 1.25 times the desired final concentration, and then mixing this solution with an interferon solution of 2.42 mg concentration. / ml.
• the volume of the protein solution is determined by the choice of the ratio of the polymer concentration to the target protein concentration.
• the adjustments of concentrations and pH are carried out by adding NaCl and sodium hydroxide solution.
• Example S Measurement of the average hydrodynamic diameter of the nanoparticles of different PO polymers according to the invention.
• the average hydrodynamic diameter of the PO polymer particles according to the invention is measured according to the procedure Md defined below.
• the PO solutions are prepared at concentrations of 1 or 2 mg / ml in 0.15M NaCl medium and left stirring for 24 h. These solutions are then filtered over 0, 8-0.2 ⁇ m, before analyzing them in dynamic light scattering using a Brookhaven-type apparatus, operating with a laser beam of wavelength 488 nm and vertically polarized.
• the hydrodynamic diameter of the polymer nanoparticles PO is calculated from the autocorrelation function of the electric field by the cumulant method, as described in the "Surfactant Science Series" volume 22, Surfactant Solutions, Ed. R. Zana, chap. 3, M. Dekker, 1984. The following results are obtained for polymers PO P2 P3 P4 and P6 of Example 2:
• a solution of 25 mM phosphate buffer is prepared from NaH 2 PO powder.
• a colloidal suspension of PI polymer nanoparticles is prepared by overnight dissolving the lyophilized polymer at 5 mg / ml in the previous phosphate buffer solution.
• a stock solution of BSA (Sigma A-2934) is prepared by dissolving protein at 10 mg / ml for two hours in the same buffer.
• Mixtures are made by adding predetermined volumes of the two stock solutions and dilution into the phosphate buffer so as to ultimately have a range of samples having a constant polymer concentration (0.1 mg / ml) and increasing concentrations of protein. (0 to 1, 8 mg / ml).
• the analyzes are carried out on an Ag ilent G16000A apparatus provided with a fused silica bubble capillary (type GI 600 -62-232).
• the height of the first plateau corresponding to the free protein makes it possible to determine the concentration of non-associated BSA.
• the protein is associated with the polymer nanoparticles.
• the GI test is applied to IFN formulations associated with polymers P1 to P6 of Examples 1 and 2.
• the protein concentrations of these formulations are reported in the table below.
• the measurement of the relaxation time of formulations in the presence of BSA (concentration 30 mg / ml) is carried out according to the procedure of the GI test. Concentration Critical Cl, for which the relaxation time exceeds ls is reported in Table 3 for IFN.
• Example 8 Pharmacokinetics of IFN in dogs after subcutaneous injection of an IFN formulation belonging to the selection according to the invention.
• a formulation (A) of IFN (concentration 0.3 mg / ml) and of amphiphilic polymer PI at a concentration of 30 mg / ml is prepared according to the procedure described in Example 4.
• Plasma concentration of IFN is measured on these samples by ELIS A assay (IM 3193 immunotech kit).
• FIG. 1 An average plasma concentration profile is thus obtained as shown in FIG. 1 which clearly demonstrates the prolonged release of the protein in the serum compared with a control formulation (D) outside the invention of IFN (concentration 0.3 mg / ml) and P6 amphiphilic polymer at a concentration of 40 mg / ml (see Table 5, Example 9).
• the prolongation of the release of IFN by the formulations according to the invention is estimated by measuring: (a) the time Tmax, median of the time for which the plasma concentration is maximal, (b) the time T50, the average time after which the area under the plasma concentration curve reaches 50% of its maximum measured value.
• Formulation A has a higher polymer concentration than the Cl gelling concentration measured in Example 6. In other words, the relaxation time measured in the GI test is greater than 1 second.
• This formulation A therefore belongs to the selection according to the invention.
• formulations B, C and D have concentrations lower than their gelling concentrations and do not belong to the selection according to the invention. These formulations are injected at a dose of 60 ⁇ g / kg into Beagle dogs. Plasma samples are taken at times 1, 5, 11, 24, 36, 48, 72, 96, 120, 144, 168 and 240 hours. The plasma concentration of IFN is measured as in the previous example.
• formulation A which belongs to the selection according to the invention, has a considerably increased release time compared to formulations B, C and D which do not belong to the selection according to the invention.
• Example 10 Observation of in vivo gelling of the formulations according to the invention after subcutaneous injection.
• Formulation A Isotomous aqueous solution at pH 7.3 of the polymer P6 of Example 2 concentrated at 45 mg / ml.
• Formulation B Isotonic aqueous solution at pH 7.3 of the PI polymer of Example 1 concentrated to 20 mg / ml.
• the injected sites were taken 72 hours after administration. Histological examination reveals the presence of a gelled polymer deposit for formulation B. It is in the form of uniformly colored plaques. This phenomenon is however not observed for the formulation A for which the polymer is infiltrated between the collagen fibers.
• polymer matrix B is perfectly biodegradable because the tissue has completely returned to its normal state after 21 days.

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